Summary of Work: The goals of this project are to investigate the role of altered ion homeostasis and ion signaling in cell injury and protective adaptation. We previously demonstrated that an increase in cytosolic free [Ca2+] (Cai) is causally involved in cell injury, and that reducing the rise in Cai is protective. This project has two lines of investigation. One series of studies are investigating the role of Na/Ca exchange in cell injury using transgenic mice that overexpress the Na/Ca exchanger. These studies have shown that perfused hearts from male, but not female Na/Ca overexpressor mice have poor recovery of function following ischemia, most likely due to an increase in Cai, because a larger proportion of the increased intracellular [Na+] which occurs during ischemia exits the cell in exchange for Ca2+. The functional recovery of hearts from bilateral ovariectomized female transgenic mice was significantly worse than that of non-ovarectomized female transgenics and similar to that of male transgenic mice, suggesting that estrogen counters the effects of overexpression of the Na/Ca exchanger. A second series of studies are investigating the mechanisms involved in protective adaptation (preconditioning). Brief intermittent periods of stress (ischemia, H2O2, osmotic stress etc.), termed preconditioning, provide protection against injury during a subsequent longer period of stress. We showed previously that preconditioning reduces the rise in Cai. Current studies involve elucidating the signaling pathways that are responsible for this reduced rise in Cai. We are investigating the role of eicosanoids (lipoxygenase metabolites and P450 metabolites), calcium futile cycling across the sarcoplasmic/endoplasmic reticulum, and the stress activated protein kinase, p38. Our working hypothesis is that preconditioning activates protein kinase C and/or p38 which leads to activation of PLA2 and accumulation of 12-lipoxygenase metabolites and EETs which alter transporter and/or ion channels and reduce cytosolic calcium. For example, 12-lipoxygenase metabolites as well as EETs are reported to activate K-channels which would secondarily reduce calcium entry. Furthermore eicosnaoids can also alter SR calcium transporters and we have evidence that cell stress is associated with increased futile cycling but preconditioned hearts do not show increased futile cycling.